Controllable injector sample dilution for a liquid chromatography system

ABSTRACT

Described are a method and a system for injecting a sample into a flow of a liquid chromatography system. The method includes combining a flow of a sample and a flow of a mobile phase to create a diluted sample in the system flow. The volumetric flow rate of the sample is controlled to be at a value that yields a desired dilution ratio for the diluted sample. The particular value at which the volumetric flow rate is maintained can be determined from the desired value of the dilution ratio and the volumetric flow rate of the mobile phase. System embodiments include a syringe that can be used to provide a sample solution at a controllable volumetric flow rate for combination with a high pressure mobile phase.

RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.14/824,440, filed Aug. 12, 2015 and titled “Controllable Injector SampleDilution for a Liquid Chromatography System,” which claims the benefitof the earlier filing date of U.S. Provisional Patent Application No.62/047,220, filed Sep. 8, 2014 and titled “Controllable Injector SampleDilution for a Liquid Chromatography System,” the entireties of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to sample injection for a liquidchromatography system. More particularly, the invention relates to amethod of actively controlling the dilution of a sample at the locationof injection into the mobile phase of a liquid chromatography system.

BACKGROUND

Purified compounds are required for testing and analysis protocolsapplied in many scientific fields. Purification of a compound involvesseparating a desired component or components from a mixture thatcontains additional components or impurities. Chromatography methods canbe applied to fractionate a mixture into separate components. In liquidchromatography, a sample containing a number of components to beseparated is injected into a system flow and directed through achromatographic column. The column separates the mixture by differentialretention into its separate components. The components elute from thecolumn as distinct bands separated in time.

A typical high performance liquid chromatography (HPLC) system includesa pump for delivering a fluid (the “mobile phase”) at a controlled flowrate and composition, an injector to introduce a sample solution intothe flowing mobile phase, a chromatographic column that contains apacking material or sorbent (the “stationary phase”), and a detector todetect the presence and amount of the sample components in the mobilephase leaving the column. When the mobile phase passes through thestationary phase, each component of the sample typically emerges fromthe column at a different time because different components in thesample typically have different affinities for the packing material. Thepresence of a particular component in the mobile phase exiting thecolumn can be detected by measuring changes in a physical or chemicalproperty of the eluent. By plotting the detector signal as a function oftime, response “peaks” corresponding to the presence and quantities ofthe components of the sample can be observed.

Preparative HPLC is a convenient way to isolate and purify a quantity ofa compound for further studies or use. Depending on the specificapplication, preparative separations can be performed using largecolumns and sample sizes, or may be performed using small columns forsmaller volume collection of components. A common distinction betweenpreparative and analytical HPLC is that for preparative HPLC, the samplecomponents are collected after purification, whereas for analyticalHPLC, the sample components are simply detected and quantified.

HPLC systems sometimes require that a sample be diluted before thesample is injected into the mobile phase flowing to the chromatographycolumn. The solvent used to dilute the sample may interfere with theability to obtain a desired chromatographic resolution. Generally, it ispreferable to keep a sample focused at the head of the chromatographiccolumn; however, strong solvents can limit the retention of the sampleand instead promote the release of the sample as it enters the column.In some separations, the result may be two chromatographic peaks and, inother separations, the sample may simply flow with the solvent throughthe column.

SUMMARY

In one aspect, a method for injecting a sample into a flow of a liquidchromatography system includes providing a flow of a mobile phase at afirst volumetric flow rate and combining a flow of a sample at a secondvolumetric flow rate and the flow of the mobile phase. The combinedflows create a diluted sample having a dilution ratio that is responsiveto the first and second volumetric flow rates. The second volumetricflow rate is controlled to a value so that the dilution ratio has apredetermined value.

In another aspect, a system for injecting a sample into a flow of aliquid chromatography system includes a syringe to dispense a sample, afirst fluid channel to conduct a mobile phase, a second fluid channel incommunication with the syringe to receive the sample, a valve and acontrol module. The valve has a first port in communication with thefirst fluid channel, a second port in communication with the secondfluid channel, and a third port. The valve has at least two states ofoperation, wherein when the valve is in a first state, the first port isin communication with the third port and wherein when the valve is in asecond state, the first and second ports are in communication with thethird port. The control module is in communication with the syringe tocontrol a volumetric flow rate of the sample dispensed from the syringe.The control module is also in communication with the valve to therebycontrol the state of operation of the valve, wherein the mobile phaseconducted through the first fluid channel is dispensed from the thirdport when the valve is in the first state and wherein the volumetricflow rate of the sample is controlled to a value to obtain apredetermined sample dilution ratio of a diluted sample dispensed fromthe third port when the valve is in the second state.

In still another aspect, a computer program product for injecting asample into a flow of a liquid chromatography system includes anon-transitory computer readable storage medium having computer readableprogram code. The computer readable program code includes computerreadable program code configured to provide a flow of a mobile phase ata first volumetric flow rate. The computer readable program code alsoincludes computer readable program code configured to combine a flow ofa sample at a second volumetric flow rate and the flow of the mobilephase, wherein the combined flows create a diluted sample having adilution ratio that is responsive to the first and second volumetricflow rates and wherein the second volumetric flow rate is controlled toa value so that the dilution ratio has a predetermined value.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further advantages of this invention may be betterunderstood by referring to the following description in conjunction withthe accompanying drawings, in which like reference numerals indicatelike elements and features in the various figures. For clarity, notevery element may be labeled in every figure. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 is a block diagram of a liquid chromatography system that can beused to practice embodiments of the method of the invention.

FIG. 2 is a block diagram of an embodiment of a system for injecting asample into a flow of a liquid chromatography system when the system isconfigured for loading a sample.

FIG. 3 is a block diagram of the system shown in FIG. 2 when the systemis configured for introducing the sample into the mobile phase of theliquid chromatography system.

FIG. 4 is a flowchart representation of an embodiment of a method forinjecting a sample into a flow of a liquid chromatography system.

DETAILED DESCRIPTION

Reference in the specification to “one embodiment” or “an embodiment”means that a particular, feature, structure or characteristic describedin connection with the embodiment is included in at least one embodimentof the teaching. References to a particular embodiment within thespecification do not necessarily all refer to the same embodiment.

The mobile phase is a solvent used to dissolve a sample and carry thesample through the stationary phase of a liquid chromatography system.As used herein, the word “sample” refers to a sample solution thatcontains the sample components to be injected into the system flow ofthe liquid chromatography system. The sample is typically made availablein a sample reservoir or sample container. The sample solution may alsoinclude a sample diluent. The mobile phase may be a gradient mobilephase in which the composition of the mobile phase changes with time.

As used herein, a solvent is sometimes referred to as a “strong solvent”or a “weak solvent” to indicate the relative elution strength of thesolvent with respect to one or more other solvents. If the mobile phaseis a strong solvent, the sample dissolved in the strong solvent willhave a greater affinity for the mobile phase than the stationary phase.A strong solvent is generally capable of dissolving a greater quantityof a sample than a weak solvent; however, with the use of a strongsolvent there may be a shorter retention time and little or no retentionof the sample on the stationary phase. In contrast, if the mobile phaseis a weak solvent, the sample dissolved in the weak solvent will have agreater affinity for the stationary phase than the mobile phase. As aresult, sample components are better retained on the stationary phaseand have longer retention times. By way of non-limiting examples forreversed phase chromatography, solvents composed primarily of methanol,acetonitrile, ethanol, isopropanol or tetrahydrofuran are typicallyconsidered strong solvents whereas water is generally considered a weaksolvent. By way of non-limiting examples for normal phase chromatographyand supercritical fluid chromatography, hexane and heptane are generallyconsidered weak solvents, and methanol, ethanol and water are typicallyconsidered strong solvents.

In order for the stationary phase in the chromatographic column topreferentially retain sample components, the mobile phase is initiallycomposed of a weak solvent of sufficiently low or moderate strength toprevent the sample components from simply passing through the columnwith little or no retention or separation. A gradient mobile phasegradually increases in strength over time to elute the sample componentsat different times.

In various applications, the sample may be provided in a solution thatincludes a strong diluent which may interfere with retention of thesample components on the stationary phase. To enable the samplecomponents to be retained, or “focused,” at the head of thechromatographic column, it is often desirable to further dilute thesample solution using a weaker solvent although the volume of thediluted sample is thereby increased. The use of the phrase “dilutionratio” is used herein to describe the degree, or amount, of the dilutionand means a unit volume contribution of the sample with respect to thenumber of unit volumes contributed by the one or more diluents.

In brief overview, the invention relates to a method and a system forinjecting a sample into a flow of a liquid chromatography system. Themethod may be used to improve sample loading for a preparative system orto improve chromatographic resolution in an analytical system. Themethod includes combining a flow of a sample and a flow of a mobilephase to create a diluted sample in the system flow. The volumetric flowrate of the sample is controlled to be at a value that results in adesired dilution ratio for the diluted sample. The particular value atwhich the volumetric flow rate is maintained can be determined from thedesired value of the dilution ratio and the volumetric flow rate of themobile phase. Various embodiments of the system are based on controllingthe volumetric flow rate of a sample solution that is dispensed from asample syringe and combined with the high pressure mobile phase.

Embodiments of the system described herein allow for controlled dilutionof a sample at the location of injection into a pressurized system flow.These embodiments can be used to replace systems that are more complexand have more components. Another advantage is the wide continuous rangeof dilution ratios that can be realized. The user can reduce or minimizedilution to ensure a tight injection band. Alternatively, the user canuse a greater dilution to control the negative effects onchromatographic separations that can occur when using a strong solventdiluent at the sample source. For example, a greater dilution may bepreferred when the sample is provided in a strong solvent that wouldotherwise reduce or eliminate retention of the sample components at thehead of the chromatographic column. Conventionally, samples provided instrong solvents are further diluted by a manual addition of a seconddiluent using a second sample reservoir or container. The ability todilute the sample at the location of injection into the system flowaccording to the embodiments described herein eliminates the need forthis manual dilution preparation step and avoids the associatedpotential for human error. Still another advantage is that no changes inthe physical components of the system are required to change thedilution ratio. Instead, a user programs a sequence or issues commandsthrough a user interface to change the volumetric flow rate from thesample syringe.

The present teaching will now be described in more detail with referenceto embodiments thereof as shown in the accompanying drawings. While thepresent teaching is described in conjunction with various embodimentsand examples, it is not intended that the present teaching be limited tosuch embodiments. On the contrary, the present teaching encompassesvarious alternatives, modifications and equivalents, as will beappreciated by those of skill in the art. Those of ordinary skill havingaccess to the teaching herein will recognize additional implementations,modifications and embodiments, as well as other fields of use, which arewithin the scope of the present disclosure as described herein.

FIG. 1 is a block diagram of a liquid chromatography system 10 that canbe modified to practice embodiments of the method of the invention. Thesystem 10 includes a system processor 12 (e.g., microprocessor andcontroller) in communication with a user interface device 14 forreceiving input parameters and displaying system information to anoperator. The system processor 12 communicates with a solvent manager 16which provides one or more solvents for a mobile phase. For example, thesolvent manager 16 may provide a gradient mobile phase. A sample from asample source 20 is injected into the mobile phase upstream from achromatographic column 22 at an injection valve 24. The sample source 20can be a sample reservoir such as a vial or other container that holds avolume of the sample. In some instances, the sample source 20 provides adiluted sample that includes the sample and a diluent. Thechromatographic column 22 is coupled to a detector 26 which provides asignal to the system processor 12 that is responsive to variouscomponents detected in the eluent from the column 22. After passingthrough the detector 26, the system flow exits to a waste port; however,when used for fraction collection, a diverter valve 30 is used to directthe system flow to one or more collection vessels 28.

FIG. 2 is a block diagram of an embodiment of a system 40 for injectinga sample into a flow of a liquid chromatography system according to theinvention. The system 40 includes a valve 42 having three ports 46A, 46Band 46C (generally 46), a sample syringe 44, a sample needle 48, aninjection port 50, a needle drive 52, a sample source 54 and a controlmodule 56.

The sample syringe 44 is adapted for direct fluidic communication withthe pressurized system flow of the liquid chromatography system. Forexample, the sample syringe 44 may be formed as a stainless steel barreland plunger, and have a stroke that can be accurately controlled forspeed and displacement while operating under high system pressures(e.g., 7,000 psi).

One of the ports 46A on the valve 42 communicates with a solvent managerthrough tubing or another form of conduit to receive a system flow inthe form of a mobile phase. A second port 46B is in communication withthe injection port 50 and is configured to receive a sample to beinjected into the system flow. A third port 46C is in communication witha chromatographic column through tubing or other conduit. The valve 42is operable in at least two states in which the configurations of liquidflowing through the vale 42 are different. The valve 42 is shown in afirst state in which the mobile phase from solvent manager received atport 46A passes through the valve 42 and exits at port 46C. In a secondstate, the flow of the mobile phase through the valve 42 remains thesame; however, the sample provided at port 46B is combined with themobile phase received at port 46A such that the combined flows exit thevalve 42 at port 46C.

The injection valve 42 operates at high system pressures, for example,pressures that may exceed 25,000 psi. In some embodiments, the valve 42is a rotary shear seal valve. Examples of other types of valves that canoperate at sufficiently high pressure and be used as an injection valveinclude a gate valve, a butterfly valve, a needle valve or a rotarycylinder valve. Although shown as only having three ports 46, it shouldbe recognized that a valve having more than three ports can also be usedas long as the valve can be configured to provide the same flow pathsand switching capability as the illustrated valve 42.

Control of the injection valve 42 is achieved with the control module 56and includes the ability to switch the valve 42 between the two states.In various embodiments, the control module 56 may be implemented in asystem processor 12 (FIG. 1). In alternative embodiments, the controlmodule 56 is a computer (e.g., personal computer (PC)) or standalonecontroller that can receive commands or one or more control signals fromthe system processor 12 or other module.

The needle drive 52 is used to move the sample needle 48 to variouspositions within the system 40. For example, the needle drive 52 is usedto move the tip of the sample needle 48 into position in the samplesource 54 (e.g., sample reservoir or sample vial), as illustrated, andcan be used to move the sample needle 48 to a position in which thesample needle 48 engages the injection port 50 as shown in FIG. 3.

The control module 56 is also in communication with the sample syringe44 and is used to control the loading of sample from the sample source54 into the sample needle 48 and the dispensing of sample from thesample needle 48 into the mobile phase. Sample dilution at the locationof injection according to embodiments of the methods and systemsdescribed herein generally refers to dilution of the liquid from thesample source by the mobile phase of the chromatography system. If thesample source provides an undiluted sample, the mobile phase is the onlydiluent. In contrast, if the sample source 54 provides the sample in asample diluent, the mobile phase is a second diluent that is used tofurther dilute the raw sample. The control module 56 is used toaccurately control the volumetric flow rate of the sample dispensed fromthe sample needle 48 into the mobile phase.

In various embodiments described herein, the dilution of the sampledispensed from the sample needle 48 into the mobile phase is determinedaccording to the volumetric flow rates of the sample and the mobilephase. It will be recognized that the sample from the sample source maybe in a sample diluent and thus there may be a different dilution ratiowhich is associated with the sample at the sample source 54. The totaldilution of the sample is affected by any dilution at the sample source54 as well as the dilution achieved during injection into the mobilephase.

The illustrated system is shown with a source 60 of a wash solvent thatis in communication with the injection port 50 through a wash pump 62and a wash valve 64. These components are used to wash the fluid path inthe injection port 50 and to purge the solvent from the sample needle48. These operations may be used after completion of a sample injectionor before initiating a subsequent sample injection in order to reduce oreliminate cross-contamination that may occur between sample injectionsfor consecutive separations.

Referring to FIG. 2 and also to the flowchart representation of anembodiment of a method 200 for injecting a sample into a flow of aliquid chromatography system as shown in FIG. 4, the sample syringe 44is loaded (step 210) by acquiring sample from the sample source (e.g.,sample reservoir) 54. The volume of sample acquired is determined by theintake stroke of the syringe 44. A flow of mobile phase at a firstvolumetric flow rate is provided (step 220) through the system path ofthe liquid chromatography system in advance of and during the sampleinjection. The needle drive 52 removes the sample needle 48 from thesample source 54 and moves it into the injection port 50 as shown inFIG. 3. Subsequently, the sample syringe 44 is activated by the controlmodule 56 to dispense a flow of the sample into the mobile phase. Thiscombination (step 230) of the two flows creates a diluted sample in themobile phase. The volumetric flow rate of the sample dispensed from thesyringe 44 is controlled to a value that enables the dilution of thesample caused by the combination of the two flows to have the desireddilution ratio. For example, if the volumetric flow rate of the mobilephase is ten times the volumetric flow rate of the sample, the resultingdilution ratio is 1:10 for the liquid dispensed from the sample needle48. As described above, the total dilution for the raw sample may begreater if the sample reservoir 54 containing the sample also contains adiluent.

The control module 56 can be configured to receive data indicating thedesired volumetric flow rate for the sample, for example, from inputdata provided through a user input device. Alternatively, the user mayinput a desired dilution ratio at the user input device and the controlmodule 56 (or a processor (not shown) in communication with the controlmodule 56) can determine the corresponding volumetric flow rate for thesample and generate the appropriate signals to control the samplesyringe 44.

As will be appreciated by one skilled in the art, aspects of the presentinvention may be embodied not only as a system or a method, as describedabove, but also as a computer program product. Accordingly, aspects ofthe present invention may also take the form of an embodiment combiningsoftware and hardware aspects that may all generally be referred to as asystem, and as an entirely software embodiment (including firmware,resident software, micro-code, etc.). Furthermore, aspects of thepresent invention may take the form of a computer program productembodied in one or more computer readable mediums having computerreadable program code embodied thereon.

Any combination of one or more computer readable mediums may beutilized. The computer readable medium may be a computer readablestorage medium. A computer readable storage medium may be, for example,but not limited to, an electronic, magnetic, optical, electromagnetic,infrared, or semiconductor system, apparatus, or device, or any suitablecombination of the foregoing. More specific but non-exhaustive examplesof the computer readable storage medium include the following: anelectrical connection having one or more wires, a portable computerdiskette, a hard disk, a random access memory (RAM), a read-only memory(ROM), an erasable programmable read-only memory (EPROM or Flashmemory), an optical fiber, a portable compact disc read-only memory(CD-ROM), an optical storage device, a magnetic storage device, or anysuitable combination of the foregoing. More generally, as used herein acomputer readable storage medium may be any tangible medium that cancontain, or store a program for use by or in connection with aninstruction execution system, apparatus or device.

Computer program code for carrying out operations for aspects of thepresent invention, for example, aspects of the method embodimentsdescribed above, may be written in any combination of one or moreprogramming languages. The program code may execute entirely on theuser's computing system, partly on the user's computing system, as astand-alone software package, partly on the user's computing system andpartly on a remote computing system or entirely on the remote computingsystem or server. In the latter scenario, the remote computing systemmay be connected to the user's computing system through any type ofnetwork, including a local area network (LAN) or a wide area network(WAN), or the connection may be made to an external computer (forexample, through the Internet using an Internet Service Provider).

While the invention has been shown and described with reference tospecific embodiments, it should be understood by those skilled in theart that various changes in form and detail may be made therein withoutdeparting from the spirit and scope of the invention as recited in theaccompanying claims.

What is claimed is:
 1. A method for injecting a sample into a flow of aliquid chromatography system, the method comprising: providing a flow ofa mobile phase at a first volumetric flow rate; and combining a flow ofa sample at a second volumetric flow rate and the flow of the mobilephase, wherein the combined flows create a diluted sample having adilution ratio that is responsive to the first and second volumetricflow rates and wherein the first volumetric flow rate is controlled to avalue so that the dilution ratio has a predetermined value.
 2. Themethod of claim 1 wherein the sample includes a sample diluent.
 3. Themethod of claim 2 wherein the dilution ratio is responsive to a dilutionof the sample by the sample diluent and a dilution of the sample by themobile phase.
 4. The method of claim 2 wherein the sample diluentcomprises a solvent that is capable of dissolving a greater quantity ofthe sample than the mobile phase.
 5. The method of claim 1 wherein themobile phase is a gradient mobile phase.
 6. The method of claim 1wherein the combining of the flow of the sample and the flow of themobile phase occurs for a predetermined duration.
 7. The method of claim1 further comprising acquiring the sample from a sample source prior tocombining the flow of the sample and the flow of the mobile phase.
 8. Acomputer program product for injecting a sample into a flow of a liquidchromatography system, the computer program product comprising: anon-transitory computer readable storage medium having computer readableprogram code embodied therewith, the computer readable program codecomprising: computer readable program code configured to provide a flowof a mobile phase at a first volumetric flow rate; and computer readableprogram code configured to combine a flow of a sample at a secondvolumetric flow rate and the flow of the mobile phase, wherein thecombined flows create a diluted sample having a dilution ratio that isresponsive to the first and second volumetric flow rates and wherein thefirst volumetric flow rate is controlled to a value so that the dilutionratio has a predetermined value.
 9. The computer program product ofclaim 8 wherein the computer readable program code further comprisescomputer readable program code configured to acquire the sample from asample source prior to combining the flow of the sample and the flow ofthe mobile phase.